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. 2016 Apr 22;5(4):e003075.
doi: 10.1161/JAHA.115.003075.

Endothelial Notch1 Is Required for Proper Development of the Semilunar Valves and Cardiac Outflow Tract

Sara N Koenig  1 , Kevin Bosse  1 , Uddalak Majumdar  1 , Elizabeth M Bonachea  2 , Freddy Radtke  3 , Vidu Garg  4
Affiliations

Endothelial Notch1 Is Required for Proper Development of the Semilunar Valves and Cardiac Outflow Tract

Sara N Koenig et al. J Am Heart Assoc. .

Abstract

Background: Congenital heart disease is the most common type of birth defect, affecting ≈2% of the population. Malformations involving the cardiac outflow tract and semilunar valves account for >50% of these cases predominantly because of a bicuspid aortic valve, which has an estimated prevalence of 1% in the population. We previously reported that mutations in NOTCH1 were a cause of bicuspid aortic valve in nonsyndromic autosomal-dominant human pedigrees. Subsequently, we described a highly penetrant mouse model of aortic valve disease, consisting of a bicuspid aortic valve with thickened cusps and associated stenosis and regurgitation, in Notch1-haploinsufficient adult mice backcrossed into a Nos3-null background.

Methods and results: Here, we described the congenital cardiac abnormalities in Notch1(+/-);Nos3(-/-) embryos that led to ≈65% lethality by postnatal day 10. Although expected Mendelian ratios of Notch1(+/-);Nos3(-/-) embryos were found at embryonic day 18.5, histological examination revealed thickened, malformed semilunar valve leaflets accompanied by additional anomalies of the cardiac outflow tract including ventricular septal defects and overriding aorta. The aortic valve leaflets of Notch1(+/-);Nos3(-/-) embryos at embryonic day 15.5 were significantly thicker than controls, consistent with a defect in remodeling of the semilunar valve cushions. In addition, we generated mice haploinsufficient for Notch1 specifically in endothelial and endothelial-derived cells in a Nos3-null background and found that Notch1(fl/+);Tie2-Cre(+/-);Nos3(-/-) mice recapitulate the congenital cardiac phenotype of Notch1(+/-);Nos3(-/-) embryos.

Conclusions: Our data demonstrate the role of endothelial Notch1 in the proper development of the semilunar valves and cardiac outflow tract.

Keywords: Notch1 signaling; bicuspid aortic valve; cardiovascular genetics; congenital heart defect; conotruncal heart defects.

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Figures

Figure 1
Figure 1
Notch1 +/− ;Nos3 −/− mice display perinatal lethality. Compound mutant mice suffered ≈65% lethality by P10, as shown in (A). Nevertheless, compared with Nos3 +/− (n=18) (B), Nos3 −/− (n=20) (C), and Nos3 +/− ;Notch1 +/− (n=18) (D) littermates, Notch1 +/− ;Nos3 −/− mice did not display any embryonic lethality (A) or growth retardation at E18.5 (n=17) (E). Compared with controls (F through H), examination of E18.5 hearts revealed right ventricle enlargement in compound mutants (I). Scale bars, 2 mm. E indicates embryonic day; P, postnatal day.
Figure 2
Figure 2
Notch1 +/− ;Nos3 −/− embryos display cardiac outflow tract and semilunar valve malformations. Compared with Nos3 +/− control mice (A), compound mutant mice (B through D) displayed AoV thickening (B, arrowhead), VSD with overriding Ao (C, asterisk), and PV thickening (D, arrow), as summarized in the table (E). Nos3 +/−, n=7; Nos3 −/−, n=7; Notch1 +/− ;Nos3 +/−, n=7; Notch1 +/− ;Nos3 −/−, n=9. Scale bars=200 μm. *P<0.05. Ao indicates aorta; AoV, aortic valve; LV, left ventricle; PA, pulmonary artery; PV, pulmonary valve; RV, right ventricle; VSD, ventricular septal defect.
Figure 3
Figure 3
Notch1 +/− ;Nos3 −/− embryos display abnormal aortic valve remodeling. Compared with Nos3 +/− (A), Nos3 −/− (B), and Nos3 +/− ;Notch1 +/− (C) animals at embryonic day 18.5, compound mutant mice demonstrated thickened aortic valves (D), as summarized in (K) (Nos3 +/−: n=5, median 0.01 [IQR 0.01–0.01125]; Nos3 −/−: n=5, median 0.01 [IQR 0.01–0.01]; Notch1 +/− ;Nos3 +/−: n=5, median 0.015 [IQR 0.01–0.02]; Notch1 +/− ;Nos3 −/−: n=5, median 0.0225 [IQR 0.02–0.03]). Notch1 +/− ;Nos3 −/− animals also display a reduced number of nuclei per unit area (J and L) compared with Nos3 −/− animals (I) and controls (L) (Nos3 +/−: n=5, median 17 200 [IQR 15 675–19 750]; Nos3 −/−: n=5, median 18 000 [IQR 14 975–20 375]; Notch1 +/− ;Nos3 +/−: n=5, median 14 600 [IQR 14 350–16 950]; Notch1 +/− ;Nos3 −/−: n=5, median 10 400 [IQR 9000–11 767]). Notch1 +/− ;Nos3 −/− mice were found to have thickened aortic valve leaflets (arrowhead) at embryonic day 15.5 (H) compared with Nos3 +/− (E), Nos3 −/− (F) and Notch1 +/− ;Nos3 +/− (G) animals, accompanied by a decrease in aortic valve excavation (M) (Nos3 +/−: n=4, median 23.90 [IQR 18.80–33.60]; Nos3 −/−: n=4, median 25.10 [IQR 17.30–37.80]; Notch1 +/− ;Nos3 +/−: n=4, median 29.80 [IQR 24.00–32.80]; Notch1 +/− ;Nos3 −/−: n=4, median 16.80 [IQR 10.40,– 20.05]). Scale bars=200 μm. *P<0.05; **P<0.01. IQR shows the 25th and 75th percentiles. Ao indicates aorta; IQR, interquartile range; LA, left atria; RV, right ventricle.
Figure 4
Figure 4
Endothelial‐specific Notch1 haploinsufficiency in a Nos3‐null background recapitulates the Notch1 +/− ;Nos3 −/− cardiac phenotype. Notch1 fl/wt;Tie2‐Cre +/−;Nos3 −/− embryos (n=6) were found to have thickened aortic and pulmonary valve leaflets (C and D) at embryonic day 18.5 compared with Notch1 fl/wt ;Tie2‐Cre −/− ;Nos3 −/− animals (n=4) (A and B). E, Table showing cardiac phenotypes in Notch1 fl/wt;Tie2‐Cre +/−;Nos3 −/− embryos, which also displayed VSD and overriding Ao. F, Reduced AoV cell density in Notch1 fl/wt;Tie2‐Cre +/−;Nos3 −/− embryos (n=5, median 12 196 [IQR 9769–14 446]) compared with Notch1 fl/wt ;Tie2‐Cre −/− ;Nos3 −/− embryos (n=4, median 17 100 [IQR 16 745–21 473]). Scale bar=200 μm. *P<0.05; **P<0.01. IQR shows the 25th and 75th percentiles. Ao indicates aorta; AoV, aortic valve; IQR, interquartile range; ns, not significant (P=1.00); PV, pulmonary valve; RV, right ventricle; VSD, ventricular septal defect.
Figure 5
Figure 5
SHF‐specific Notch1 haploinsufficiency in a Nos3‐null background partially recapitulates the Notch1 +/− ;Nos3 −/− cardiac phenotype. Notch1 fl/wt ;Mef2C‐Cre +/− ;Nos3 −/− embryos (n=7) were found to have thickened AoV and PV leaflets (C and D) at embryonic day 18.5 compared with Notch1 fl/wt ;Mef2C‐Cre −/− ;Nos3 −/− animals (n=4) (A and B). Notch1 fl/wt; Mef2C‐Cre +/−;Nos3 −/− mice also displayed VSDs and overriding Ao (E), although to a lesser extent than what is seen in the Notch1 +/− ;Nos3 −/− mice, which is also observed by a reduction in AoV cell density in Notch1 fl/wt;Mef2C‐Cre +/−;Nos3 −/− embryos (n=3, median 10 455 [IQR 10 144–11 442]) compared with Notch1 fl/wt ;Mef2C‐Cre −/− ;Nos3 −/− animals (n=3, median 13 258 [IQR 11 271–14 512]) (F). Lineage tracing with ROSA26 mT/mG;Mef2C‐Cre +/− mice (n=4) revealed SHF‐derived endothelial cells (arrowheads) lining the AoV and aortic root (G and H). GFP indicates SHF‐derived cells; PECAM1 indicates endothelial cells. Scale bars=100 μm. **P<0.01. IQR shows the 25th and 75th percentiles. Ao indicates aorta; AoV, aortic valve; GFP, green fluorescent protein; IQR, interquartile range; ns, not significant (P=1.00); LV, left ventricle; PV, pulmonary valve; RV, right ventricle; SHF, second heart field; VSD, ventricular septal defect.
Figure 6
Figure 6
Neural crest–specific Notch1 haploinsufficiency in a Nos3‐null background does not recapitulate the cardiac phenotype of the Notch1 +/− ;Nos3 −/− mice. Notch1 fl/wt ;Nos3 +/− mice were bred to Notch1 wt/wt ;Wnt1‐Cre +/− ;Nos3 −/− male mice, creating neural crest–specific Notch1 haploinsufficiency in a Nos3 −/− background. Notch1 fl/wt;Wnt1‐Cre +/− ;Nos3 −/− embryos (n=4) were found to have normal AoV and PV leaflets (A through D) at embryonic day 18.5 compared with Notch1 fl/wt ;Wnt1‐Cre −/− ;Nos3 −/− animals (n=4) (E). There was no evidence of VSDs and overriding Ao in Notch1 fl/wt; Wnt1‐Cre +/−;Nos3 −/− mice (E). Scale bar=200 μm. Ao indicates aorta; AoV, aortic valve; ns, not significant (P=1.00); LV, left ventricle; PV, pulmonary valve; RV, right ventricle; VSD, ventricular septal defect.
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